Neuroradiological, genetic and clinical characteristics of histone H3 K27-mutant diffuse midline gliomas in the Kansai Molecular Diagnosis Network for CNS Tumors (Kansai Network): multicenter retrospective cohort

This study aims to elucidate the clinical and molecular characteristics, treatment outcomes and prognostic factors of patients with histone H3 K27-mutant diffuse midline glioma. We retrospectively analyzed 93 patients with diffuse midline glioma (47 thalamus, 24 brainstem, 12 spinal cord and 10 other midline locations) treated at 24 affiliated hospitals in the Kansai Molecular Diagnosis Network for CNS Tumors. Considering the term “midline” areas, which had been confused in previous reports, we classified four midline locations based on previous reports and anatomical findings. Clinical and molecular characteristics of the study cohort included: age 4–78 years, female sex (41%), lower-grade histology (56%), preoperative Karnofsky performance status (KPS) scores ≥ 80 (49%), resection (36%), adjuvant radiation plus chemotherapy (83%), temozolomide therapy (76%), bevacizumab therapy (42%), HIST1H3B p.K27M mutation (2%), TERT promoter mutation (3%), MGMT promoter methylation (9%), BRAF p.V600E mutation (1%), FGFR1 mutation (14%) and EGFR mutation (3%). Median progression-free and overall survival time was 9.9 ± 1.0 (7.9–11.9, 95% CI) and 16.6 ± 1.4 (13.9–19.3, 95% CI) months, respectively. Female sex, preoperative KPS score ≥ 80, adjuvant radiation + temozolomide and radiation ≥ 50 Gy were associated with favorable prognosis. Female sex and preoperative KPS score ≥ 80 were identified as independent good prognostic factors. This study demonstrated the current state of clinical practice for patients with diffuse midline glioma and molecular analyses of diffuse midline glioma in real-world settings. Further investigation in a larger population would contribute to better understanding of the pathology of diffuse midline glioma. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-024-01808-w.

Essential information about DMG has been summarized in the WHO Blue Book [57].Even after CNS WHO 2021, however, several researchers have reported additional findings [5,6,23,27,30,32,36,53,54,58,62].Owing to its rarity, however, there are few comprehensive reports and there are remaining inconsistencies about DMG.There are major concerns regarding prediction of clinical behavior and outcomes in daily practice; there is a lack of real-world data on clinical and molecular characteristics and treatment outcomes.The current study investigates the prevalence and impact of previously-reported biomarkers.
For the present study, we reviewed the inclusion criteria of DMG used in previous reports that focused upon the midline structures.We collected histone H3 K27Mmutant diffuse gliomas at the midline location in the Kansai Molecular Diagnosis Network for CNS Tumors (Kansai Network) cohort.This is a multi-institutional retrospective cohort study of 93 cases of DMG treated at 24 hospitals in the Kansai Network.We aim to elucidate both clinical and pathological features of cases of DMG, as well as treatment outcomes and prognostic factors of patients with DMG in real-world settings.

Ethics
This study was carried out in accordance with the principles of the Declaration of Helsinki.The study was approved by the Institutional Review Board (IRB) of Osaka National Hospital (No. 713), Wakayama Medical University (No. 98), Wakayama Rosai Hospital (No. 20 , and all collaborating institutions.Written informed consent was obtained from all patients.

Patient population and study design
This study included patients with histone H3-mutated gliomas who were treated at one of 27 institutions or hospitals participating in the Kansai Network [41].Between May 2007 and July 2022, we collected a total of 4128 samples including all kinds of primary and recurrent gliomas from 72 institutions.From this databank, we focused on diffuse gliomas with histone H3 mutation and collected 118 cases (116 cases with H3F3A mutation and two cases with HIST1H3B mutation).Among the cases with H3F3A mutation, 107 cases had the K27M mutation, and nine cases had the G34R/V mutation.In this study, we examined 109 cases from 24 institutions, consisting of 107 cases with the K27M mutation and two cases with the HIST1H3B mutation.Patient selection is summarized in a flowchart in Fig. 1.Diagnosis of diffuse gliomas was initially confirmed by histopathological examination at each institution or hospital.

Tumor location (Kansai classification)
Preoperative images were available in 106 of the 109 cases (the anatomic tumor locations were identified by medical records in three cases).Neuroradiological assessments were performed by three experienced board-certified neurosurgeons (N.H., H.N., H.K.) and three additional senior board-certified neurosurgeons (J.F., K.M., Yo.Ka.) to reach a consensus.Tumor locations in this study were determined using the anatomical criteria as follows: • The main anatomical structure in which the tumor is solely located is defined as the tumor location, for example, the thalamus, the brainstem, the spinal cord, etc. (Additional file 4: Figure S1A).• If tumors were distributed across multiple anatomical regions in a contiguous manner, the presumed tumor origin site was determined based on the loca-tion of contrast-enhanced lesions and the progression pattern of FLAIR high-signal areas (Additional file 4: Figure S1B).• The cases in which non-contiguous multifocal tumors were detected and in which the main anatomical structure of the tumor could not be determined were defined as unclassified.For example, a case might equally harbor both the thalamus and the corpus callosum (Additional file 4: Figure S1C, D).
To discriminate between the "midline" and "non-midline" locations for this study, we applied the following criteria: • The thalamus, brainstem, spinal cord, pineal gland, hypothalamus, cerebellum, and ventricles were categorized as midline, and the basal ganglia and corpus callosum (as part of the cerebral hemisphere) were categorized as non-midline [45,49,50] (Table 1).• If a tumor was located at the basal ganglia or corpus callosum but mainly involved midline structures such as the thalamus or the brainstem, it was categorized as a midline tumor (Additional file 4: Figure S1C, E, Table 1).• If a tumor mainly involved the cerebral hemisphere, it was categorized as a non-midline tumor (Additional file 4: Figure S1F, Table 1).

Clinical information
Clinical information was collected from medical records including patient demographics, preoperative Karnofsky performance status (KPS) scores, the extent of surgical resection (EOR), adjuvant radiation and chemotherapy

Histopathological examination
All cases were subject to central pathology review by a senior board-certified neuropathologist (Yo.Ko).Histological diagnosis was made based on the CNS WHO 2021 classification [57].

Results
Preoperative imaging analysis resulted in 93 of 109 cases being categorized as having midline tumors (diffuse midline tumor, DMG) (85%) and they were enrolled in this study.The other sixteen cases (15%) were categorized as having non-midline tumors.
The clinical and molecular characteristics of the 93 patients analyzed in this study are shown in Table 2. Anatomical tumor locations were classified into four groups: the thalamus group (47 cases), the brainstem group (24 cases), the spinal cord group (12 cases) and other midline locations group (10 cases) (Fig. 2a, Table 2).Other midline locations included the ventricle (two cases), the basal ganglia (two cases), and the cerebellum (2 cases), and four cases were unclassified.Cases in the basal ganglia and unclassified cases mainly involved midline locations.Distribution of the patients' age and sex are shown in Fig. 2b, and detailed information on each patient is shown as a tile panel in Fig. 3.

Clinical characteristics
There were 55 men (59%) and 38 women (41%) with a median age of 31 years (range 4-78 years).As shown in Figs.2b and 3, only 26 patients were ≤ 18 years old (28%), and just seven patients were ≥ 70 years old (8%).According to the tumor locations, there seems to be significant difference in age distribution at other midline locations vs. the thalamus, the brainstem and the spinal cord locations (p = 0.041) (Table 2).As for sex, male predominance may exist in each location, but without significant difference (p = 0.809) (Table 2).
In MR images, gadolinium (Gd) enhancement of the tumor, as a high grade imaging feature, was observed in 68 tumors (73%) (Table 2 and Fig. 3).There was significant difference between groups (p = 0.016).Notably, Gd enhancement was not observed in 10 tumors (42%) in the brainstem group, a higher proportion than in the other groups.Hemorrhage was observed to have occurred in only one case in the thalamus [32].
Based on histopathological findings including morphology, cellularity, mitotic figures, and features of glioblastoma (GBM) (microvascular proliferation or necrosis) according to CNS WHO 2021 classification [57], 40 patients (43%) had GBM features and were diagnosed as having GBM.Thirty-six patients (39%) had diffusely infiltrative gliomas with histological features of anaplasia and displayed significant mitotic activity but without microvascular proliferation or necrosis, and they were diagnosed as having high-grade glioma (HGG) without GBM features.Sixteen patients (17%) had diffusely infiltrative glioma without histological features of anaplasia and displayed no/low mitotic activity without microvascular proliferation or necrosis, and they were diagnosed as having low-grade glioma (LGG).Approximately half of the cases with GBM features were in the thalamus and spinal cord groups (55% and 50%, respectively).Meanwhile, 79% of cases with LGG or HGG without features of GBM were in the brainstem group (Table 2).Preoperative KPS scores ranged between 20 and 100 (median 70), and 46 patients had a score of ≥ 80 (49%).It may be notable that the preoperative KPS score was ≤ 70 in 67% cases in the spinal cord group.However, distribution of preoperative KPS score was not significantly different between tumor locations (p = 0.568).LGG, diffusely infiltrative glioma without histological features of anaplasia, which displays no/low mitotic activity; HGG, diffusely infiltrative glioma with histological features of anaplasia and displays significant mitotic activity; GBM features, microvascular proliferation or necrosis  Regarding EOR, 5 (5%), 11 (12%), 18 (19%), and 59 (63%) patients underwent GTR, STR, PR, and biopsy, respectively.Regardless of tumor locations, biopsy tended to be performed: it was performed in the thalamus, the brainstem, the spinal cord, and in other locations in 53%, 79%, 67% and 70% of cases, respectively (Table 1).EOR was not significantly different between tumor locations (p = 0.05).Surgical resection (46%) was more common in the thalamus group (46%) than in the other groups.
The observation period ranged between 0.5 and 63.5 months (median 15.6 months).During the observation period, tumor progression was observed in 58 patients (58/77, 75%).Repeat surgical resection was performed in seven cases (7/58, 12%).According to tumor locations, 6 of the 30 patients with a tumor in the thalamus and 1 of the 15 patients with a tumor in the brainstem underwent repeat resection [22] (Table 2, Fig. 3).

Molecular characteristics
As shown in Table 2 and Fig. 3, HIST1H3B p.K27M mutation was observed in only two cases in the thalamus (2%) and all other cases had H3F3A p.K27M mutation (98%).IDH1/2 was wild-type in all cases, regardless of the tumor location.TERT promoter mutations were observed in only three cases in the thalamus (3%).MGMT promoter methylation was found in nine cases (10%) across tumor locations: five cases in the thalamus (11%), one case in the brainstem (4%), one case in the spinal cord, and two cases in other locations (20%), but there was no statistical difference (p = 0.304).TP53 mutation was detected in approximately half of cases across tumor locations (57%); there was no statistically significant difference (p = 0.207).BRAF p.V600E was observed in only one case in the thalamus (1%).This patient had co-occurrence of H3 p.K27M and BRAF p.V600E mutations.FGFR1 mutation was found in 13 cases across tumor locations (14%), but there was no significant difference in frequency between the four locations (p = 0.619).Moreover, there was no significant difference between brainstem location (n = 24) and non-brainstem locations (n = 69) (p = 0.215).Notably, FGFR1 mutations were observed in almost all adult cases with the exception of one pediatric case in the brainstem (Fig. 3).In the cases harboring FGFR1 mutation, TP53 mutation occurred in five cases (5/13, 38%).EGFR mutation was observed in three patients (3%) (one in the thalamus, two in the brainstem).These cases can be diagnosed as DMG, EGFR-mutant, one subtype in DMG, H3 K27-altered.
As the results of univariate analysis of the relationships between characteristics and estimated survival times for all cases of DMG, female sex, preoperative KPS score of ≥ 80, adjuvant RT + TMZ treatment and RT dose (≥ 50 Gy) were significantly associated with longer OS (Table 3).
The results of multivariate analysis of factors associated with OS are also shown in Table 3. Independent factors for good prognosis in the present cohort were identified as female sex and preoperative KPS score of ≥ 80.

Discussion
For the present study, we reviewed histone H3 K27Mmutant diffuse gliomas located at the midline structures in the Kansai Network dataset.We found 93 patients with midline DMG (47 in the thalamus, 24 in the brainstem, 12 in the spinal cord, and 10 in other midline locations).A separate article will report on non-midline tumors in more detail.The results of this study could be said to be representative of the current state of clinical practice for patients with DMG and molecular analyses of DMG in real-world settings.

Tumor location
Diffuse midline glioma, H3 K27-altered is defined as a tumor found in the thalamus, brainstem, spinal cord, and occasionally in the pineal gland, the hypothalamus or the cerebellum [57].However, in clinical practice, H3 K27M-mutant diffuse gliomas could exist at the anatomically non-midline location.As shown in Additional file 1: Table S1, the definition of midline may have been confused in previous studies of DMG.For example, a tumor located at corpus callosum or basal ganglia was considered to be a midline tumor by some researchers, but as a non-midline tumor by others [1,2,7,11,19,23,25,31,39,40,42,51,55,60,62].Diffuse glioma located at the thalamus along with the basal ganglia or both the thalamus and the corpus callosum was included in the studies of DMG [25,28,56].The basal ganglia, embryologically associated with the cerebral cortex, is sometimes the location in which diffuse hemispheric glioma, H3 G34mutant arise [19,60].From developmental and anatomical points of view, the cerebrum including the corpus callosum and the basal ganglia may be usually considered as non-midline structures [45,49,50].However, a thalamic glioma involving the basal ganglia or corpus callosum would be categorized within the DMG [25,28,56].On the other hand, there are some reports of the cerebral cortex being included in the location of the DMG [31,38,42,51,55,60].As for cerebellum, the vermis is apparently located at the midline, but diffuse glioma at the cerebellar hemisphere have sometimes been classified as non-midline tumors [21].Meanwhile, a tumor in the ventricle was included in several DMG studies, although the ventricle was not described in CNS WHO 2021 [1,2,7,14,20,31,33,47,55,57,62].Additionally, one study of DMG included diffuse glioma in the suprasellar region [51,62].For diffuse glioma extending from the spinal cord to the thalamus, one report introduced the concept of 'diffuse growth along with brain axis' [14].Others have used the term 'whole-brain type lesions' for widespread lesions involving three or more contiguous lobes in the brain, and involvement of one or more traditional midline structures [39].
Based on these previous reports, we classified tumors in which the primary location was identified in the ventricles as 'other midline locations' (Table 1).Furthermore, among tumors which primarily involved the corpus callosum or basal ganglia, those which predominantly involved midline structures were classified as other midline locations, and tumors that primarily included the cerebral hemisphere were classified as non-midline, respectively (Table 1).In cases of non-contiguous, multifocal lesions where the primary location was indeterminate, we classified them as other midline locations if the main area involved midline structures, and as nonmidline if it involved the cerebral hemispheres (Table 1).Using these criteria, we excluded 16 non-midline cases of 109 patients with H3 K27M-mutant diffuse glioma in Kansai Network cohort, as described in the Material and Methods section above.However, tumor locations of DMGs are sometimes heterogenous and complicated, so it may be difficult to identify the true tumor origin.We therefore suggest one standard definition for DMGs.However, this may still be incomplete, and future validation and reconsideration will be needed using a larger cohort, which we believe will improve the understanding of the features of DMGs.

Age
DMG is categorized in the pediatric-type diffuse highgrade gliomas of CNS WHO 2021; however, DMG may occur in adults, as well as in children and adolescents [14, 24, 26, 27, 33, 35, 42-44, 51, 55, 56, 60-62].Previously, not-so-small percentages of adult cases were included in studies of DMG.For example, a recent study by Zheng et [58,62].In our study cohort, the percentage of patients aged ≥ 19 years was 72.0%, so it may be higher than that of previous studies.There may be a higher occurrence in adults compared with in children [14,56].However, it should be taken into account that the limited number of pediatric cases may be due to the lower amount of surgical tissue sampling for brainstem tumors, which are more common in children than in adults [57].DMG is generally thought to occur more commonly in children, but given the larger adult population, it is believed that the number of adult cases has become more prevalent as a result.DMG should nonetheless be considered as the differential diagnosis of adult diffuse gliomas.

Sex
Gliomas are known to have higher incidence and poorer prognosis in men [34,48].Numerous studies have indicated that women have a better prognosis than men, with factors such as hormones, metabolism, the immune system, genetic and molecular mechanisms, neurogenic niches and therapeutic responsiveness, among other factors, being suggested as reasons for this [8,48].None of the previous DMG reports found a significant difference in the prognosis by sex [23,56,62].This study is thus the first report to list female sex a favorable prognostic factor in DMG.

Histopathological characteristics
In this study, histopathological features of DMGs were varied, and diagnosed as LGG (17%), HGG without features of GBM (39%) or HGG with features of GBM (43%).[62].In this study, we observed similar microvascular proliferation (21/92, 23%), and tumor cell necrosis (28/92, 30%).These findings indicate that DMGs may show predominantly HGG or GBM histopathological features.On the other hand, some tumors showed LGG characteristics in morphology, despite their poor clinical prognosis.There might be a diagnostic limitation due to tiny biopsy specimens for DMG.Moreover, biopsies of low grade regions from tumors with high grade imaging features could be a potential confounder, especially in the biopsy cases; indeed, there were 24 cases (45%) in this study, comprising 11 cases in the thalamus group (50%), six cases in the brainstem group (31%), four cases in the spinal cord group (67%) and three cases in the others group (50%) (Additional file 3: Table S3).However, the present findings may indicate that H3 K27M-mutation does not always induce malignant histopathological phenotypes.Significance of histological malignant transformation occurring in DMGs therefore requires examination in future studies in combination with molecular analysis.

Molecular features
Regarding diagnostic molecular pathology, CNS WHO 2021 Blue Book stated that co-occurrence of histone H3 K27 mutation with IDH mutations is exceptional; correspondingly, all cases revealed IDH wildtype in our genetic analysis [57].Similarly, TERT promoter mutations and MGMT promoter methylation represent rare events in DMGs.However, TERT mutated and MGMT methylated were detected in 3% and 9% of our cases, respectively, and these were mainly in the thalamus [57].
Only one patient in our cohort (a 4-year-old girl) had bilateral thalamic tumors harboring HIST1H3B p.K27M and EGFR mutations (Additional file 4: Figure S1B).As described in the WHO Blue Book, bi-thalamic tumors are more common in the EGFR-subtype of DMGs, most often occurring during childhood, with median age of 7-8 years [57].
Histone H3 K27M mutations are generally found to be associated with collaborating mutations of canonical cancer-associated pathways [57].For example, TP53 mutations were found in 57% of our study cohort, being detected predominantly in H3.3 p.K28M (K27M)mutant and EGFR-mutant cases according to a previous report [57].BRAF p.V600E mutation co-occurred in just one case (1%) in this study with H3.3 p.K28M (K27M) mutation [57].Gain-of-function mutation and genetic amplification of growth factor receptor involved in brain development are said to be common in H3 K27Mmutant DMGs, and FGFR1 mutation was found in 14% of patients in the present study [57].A recent comprehensive genomic study of H3F3A-mutant high-grade gliomas revealed that FGFR1 hotspot point mutations (N546K and K656E) were exclusively identified in H3 K27Mmutant DMGs (64/304, 21%); these tend to occur in older patients (median age: 32.5 years) and mainly arise in the diencephalon [54,58].In this study, FGFR1 mutations were mainly observed outside of the brainstem, replicating the findings reported by Williams et al. [58].The above findings were also similar to those observed in Japanese cases, and demonstrating a similar trend.Mutations were reportedly suggested to be associated with a favorable prognosis, and FGFR1 mutations are mutually exclusive with TP53 mutation [43].TP53 mutations are associated with a poor prognosis [5,43,56].However, these trends were not observed in our study cohort; these differences in prognostic factors and variations may be attributed to racial disparities.A future study will aim to validate these points within a larger sample size.

Relevance to treatments
Standard of care for DMG has never been determined, but several treatment options have been suggested, regardless of evidence.Surgical resection of DMG is often difficult, and in our cohort, biopsy tended to be undertaken (63%).However, aggressive resection may be attempted if feasible, and there were few cases in our cohort in which GTR was actually possible (5%) [23].On the other hand, adjuvant RT + TMZ was conducted in the majority of our cohort (78%).Radiotherapy has been regarded as an important treatment option for brainstem gliomas, as is DMG [23].TMZ concomitant with and adjuvant to RT is a widely used approach to GBM, but the role in cases of DMG has never been demonstrated [12,46].In our series, BEV was administered in 57% of cases, and there is a previous report of effectivity [59].
RT has been suggested in several studies to prolong the patients' survival, although there is also a report to the contrary that radiotherapy does not influence prognosis [6,23,56].Regarding the radiation dose, a standard protocol for DMG has never been established, but it often ranges from 36 to 65 Gy [6,36,42].In our study cohort, 80% of patients received 50-60 Gy.The spinal cord group, however, was likely to receive a lower radiation dose (< 50 Gy), probably due to a spinal cord tolerance dose of < 50 Gy, and to avoid potential adverse effects such as bone marrow suppression in the long lesions.As for the prognostic impact of RT, survival benefit was demonstrated when we used ≥ 50 Gy for patients of our study.

Prognostic factors
The treatment outcomes of our series are mostly consistent with those of previous reports (Additional file 2: Table S2).To date, several prognostic factors of DMGs have been suggested (Additional file 2: Table S2).Clinical factors such as age, sex, tumor location, tumor size, EOR and radiation have been considered in some reports [6, 13-16, 23-25, 28, 37, 42-44, 53, 56, 62].As for pathological and molecular factors, there has been previous discussion of histological grading, Ki-67 labelling index, histone H3 subtype and mutations of EZH2,TP53, ATRX, TERT promoter, BRAF and FGFR1 [3, 6, 9, 13-16, 23-25, 28, 37, 42-44, 53, 56, 62].As for tumor locations, brainstem location is reportedly a poor prognostic factor [16,62], but in this cohort, there was no significant difference in OS between the four tumor location groups.Adulthood has also been reported as a good prognostic factor [43,44,54], but in this cohort, there was no significant difference in OS between adults and infants.As for sex, it was not previously reported to be a prognostic factor, but we found female sex to be an independent factor in good prognosis.Meanwhile, for pathological findings, no significant difference was found among WHO grade 2,3,4 for prognosis [62], and we obtained similar results in this study as well.As for molecular factors, EZH2 expression, TP53 mutation, ATRX expression, are reportedly poor prognostic factors and FGFR1 mutation is reportedly a good prognostic factor [24,43,56], but we found no significant difference in OS between TP53 mutations in our cohort.We did not investigate EZH2 and ATRX expression in this cohort.RT is reportedly a good prognostic factor [44,56], and similarly we found RT ≥ 50 Gy to be a good prognostic factor in this cohort.
As for the prognostic impact of each factor, however, consistent results cannot be achieved universally through studies; the limited number of study patients could partly explain the absence of statistical power to detect differences between groups.In our series, there was no statistically significant difference in OS according to age, location, resection, histological grading or genetic status (Table 3).On the other hand, our multivariate analysis identified female sex and preoperative KPS score ≥ 80 as independent prognostic factors (Table 3).Further investigation in a larger cohort could contribute to a better understanding of the prognostication of DMGs.

Summary of the present study and future challenges
Complete resection of DMGs without inducing new neurological deficits is challenging.In this study, no significant difference in OS was observed based on the resection rate, but a significant difference in OS was found based on the radiation dose.It is considered crucial to complete radiation therapy without compromising KPS through surgery as a treatment.We identified no significant prolonging of OS in cases with FGFR1 mutations, but the development of local treatment with molecular targeted drugs is desired.

Limitations
Owing to the multi-institutional retrospective cohort design, this study has several limitations.Unlike in a randomized study, there could be selection bias regarding the distribution of tumor locations and decision-making of treatment strategy.The limited number of patients could explain the lack of statistical power to detect differences between groups.Attending physicians may decide to deliver treatments with consideration of the patients' age, conditions and wishes, and thus patient selection could affect the survival findings.Variation of treatment regimen at multiple institutions, such as radiation protocol and dose schedule, should also be considered.The modest prognostic impact of clinical and molecular characteristics might be partly due to the limited population.
Our Kansai classification has limitations.The ambiguity of the current midline terminology in DMG allowed us to discriminate between the midline and non-midline structures for definition of DMG.However, it is challenging to determine the location of the origin of DMG.Some tumors which appear centered in the hemispheres have involvement of midline structures.There is the possibility, for example, of a tumor starting in the midline, but from which cells that migrated outward ultimately formed the most aggressive-appearing regions according to images.Any diffuse glioma with H3 K27M mutation would qualify for the diagnosis of DMG.Further studies could help to clarify this problem.It would nonetheless be better to consider that the Kansai classification is our approach in this study for better understanding of the pathology of DMG.
There are also limitations in this study regarding the discrimination between the midline and non-midline structures for definition of DMG.In our Kansai classification, the basal ganglia and corpus callosum were categorized as non-midline structures and were excluded from the analysis of this study, although tumors located at the basal ganglia or corpus callosum but mainly involving midline structures such as the thalamus or brainstem were categorized as midline tumors (Table 1).In the notion that any diffuse glioma with H3 K27M mutation would qualify for the diagnosis of DMG, the current midline terminology in DMG would not be necessary.Further studies of diffuse non-midline gliomas including the basal ganglia and corpus callosum tumors could help to clarify this problem.

Conclusions
Considering the term "midline" areas, which had been confused in previous reports, we classified four midline locations based on previous reports and anatomical findings in this study, and reported characteristics and outcomes of patients with histone H3 K27M-mutant DMG in the Kansai Network.This community-based study is suggested to be representative of the present status of real-world practice.Further investigation in a larger patient population could contribute to better understanding of the pathology of DMG.

Fig. 3
Fig. 3 Tile panel demonstrating clinical and molecular characteristics of histone H3 K27-mutant diffuse midline glioma patients in Kansai Network (n = 93)

Fig. 5
Fig. 5 Kaplan-Meier curves according to clinical factors: age (a), sex (b), preoperative KPS score (c), extent of surgical resection (d) adjuvant treatment (e) and radiation dose (f) in the study cohort

Table 2
Clinical and molecular characteristics of histone H3 K27-mutant diffuse midline glioma patients in Kansai Network (n = 93)